This proposal concerns the cellular, genetic and molecular basis for the formation of the prechordal plate which can act as an inductive center. The foundation for the studies originates from a mutagenesis screen of the zebrafish genome and discovering 6 loci which affect prechordal plate development or function. One of these mutants, one-eyed pinhead (oep), is required in prospective prechordal plate cells by the onset of gastrulation as defined by various molecular markers. The notochord is unaffected in the oep mutant so the gene could be involved in the segregation of the prechordal plate lineage and acts autonomously as judged from labeling studies. There are three alleles available at the locus, two ENU induced and one by irradiation. The first aim will be to determine the origin and behavior of prechordal plate cells in pregastrula wildtype and oep embryos. The question being posed is when do the prechordal plate cells become distinct from other dorsal cell types and become regionalized? Classical fate mapping will be performed by labeling single cells before gastrulation and following their lineage by time-lapse video microscopy. Additionally, labeled wildtype and oep mutant cells will be co-transplanted into unlabeled hosts and their behavior and fate will be compared. The second aim will attempt to isolate and identify the oep gene using the current methodologies available such as exon trap. The third specific aim involves the characterization of the other loci which affect prechordal development. Additional alleles will be generated at the loci with further phenotypic characterization and epistatic relationships to oep being established. Also the mutations will be placed on the zebrafish genetic map.